Degeneration of intervertebral discs due to smoking: experimental assessment in a rat-smoking model

We have investigated the intervertebral discs of rat-smoking models to demonstrate that smoking is a cause of degenerative intervertebral disc disease. A smoking box was developed for this study. We exposed 8-week-old rats to indirect tobacco smoke inhalation. Each rat was forced to inhale the smoke from one cigarette per hour. The mean blood nicotine level of rodents exposed to cigarette smoke corresponds to about twice that of ordinary human smokers. Histological and immunological studies were then performed to assess the effects of smoking for varying periods of time. After 8 weeks, the chondrocytes in the disordered annulus fibrosus layer tended to grow larger and attain a rounder form than normal chondrocytes. The interleukin-1 level in the 8-week smoking group was significantly higher than that of the control group. Tobacco smoke inhalation increased local production and release of inflammatory cytokines and resultant decomposition of chondrocyte activity.     

Passive cigarette smoking changes the circadian rhythm of clock genes in rat intervertebral discs

We aimed to elucidate the molecular changes in intervertebral discs (IVDs) caused by passive smoking. Rats were subjected to 8 weeks of passive smoking; thereafter, their lumbar vertebrae were harvested. The annulus fibrosus and cartilage endplate (AF/CEP) were harvested together, and the nucleus pulposus (NP) was isolated separately. The expression of 27,342 rat genes was analyzed. In 3 “nonsmoking” rats, 96 of 112 genes whose expression varied ≥10‐fold between the AF/CEP and NP were more highly expressed in the AF/CEP. With these differentially expressed genes, we uncovered novel AF/CEP and NP marker genes and indicated their possible novel functions. Although passive smoking induced less marked alteration in the gene expression profiles of both the AF/CEP and NP, multiple clock‐related genes showed altered expression. These genes were expressed with a circadian rhythm in IVD cells, and most genes showed a phase shift of ?6 to ?9 h induced by passive smoking. Some clock‐related genes showed abolished oscillation in the NP. Passive smoking also changed the expression levels of proteases and protease inhibitors and reduced the expression of NP marker genes. Thus, passive smoking induces changes in the circadian rhythm of a peripheral clock (IVD clock) that might be involved in molecular events related to IVD degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:39–47, 2016.

     

Discomanometry in lumbar intervertebral discs: an experimental study

Summary A new discomanometry system was developed and evaluated for application in the lumbar spine. The diagnostic reliability of discography, manometry and discomanometry (a combination of both methods) was assessed in 20 lumbar cadaver spines (95 intervertebral discs) with regard to the diagnosis of non-contained intervertebral discs. The sensitivity was 92% for manometry and 78% for discography. This indicates that 22% of the non-contained discs could not be correctly identified with common discography. The specificity was 98% for manometry and 100% for discography, indicating that contained discs could be easily identified with both methods. A combination of manometry and discography provides a high sensitivity (98%) as well as high specificity (98%). It is concluded that discomanometry is a simple, cost-effective and reliable diagnostic procedure which can precede percutaneous nucleotomy without additional risks or relevant efforts. It allows the differentiation between contained and non-contained intervertrebral discs with a high degree of diagnostic reliability in an experimental setting, which justifies its further application in patients.

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Résumé Un nouveau système de discomanométrie a été développé et évalué afin d'être appliqué au rachis lombaire. La fiabilité diagnostique de la discographie, de la manométrie et de la discomanométrie (combinaison des deux méthodes) en matière de hernie discale, a été évaluée sur 20 rachis lombaires de cadavres (95 disques intervertébraux). La sensibilité était de 92% pour la manométrie et 78% pour la discographie. Cela indique que 22% des disques présentant une hernie ne pouvaient pas être identifiés par la discographie de routine. La spécificité était de 98% pour la manométrie et 100% pour la discographie, indiquant que les disques intacts pouvaient être aisément identifiés par les deux méthodes. Une combinaison de la manométrie et de la discographie assure une grande sensibilité (98%) ainsi qu'une haute spécificité (98%). En conclusion, la discomanométrie est un procédé diagnostique simple, économique et fiable que l'on peut pratiquer avant la nucléotomie percutanée sans risques ni efforts supplémentaires importants. Elle permet de distinguer les disques intervertébraux intacts des disques rompus avec une grande fiabilité diagnostique expérimentale, ce qui justifie son utilisation chez les patients.

Presented in part at the 3rd Annual Meeting of the European Spine Society, Cambridge, September 3–5, 1992     

Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses

To study the relationships between the changes due to aging in lumbar intervertebral discs and the development of protrusion or prolapse, we carried out histological studies on operative specimens of thirty-one discs, of which twenty-two had been protruded and nine, prolapsed. The specimens were obtained during twenty-nine operations for herniation of a lumbar intervertebral disc in patients who were sixty years old or older. Changes in the anulus fibrosus were more extensive in the nine prolapsed discs than in the twenty-two protruded discs. Of the nine prolapsed discs, myxomatous degeneration, fibrosis, and swollen anular fibers were found in all nine, and cysts were seen in five. Of the twenty-two protruded discs, only five showed myxomatous degeneration; ten, fibrosis; one, a cyst; and sixteen, swollen fibers. For comparison, we also studied specimens that had been obtained at operation from twenty-one other patients, twenty to fifty-nine years old, who had a prolapsed disc. The anulus showed myxomatous degeneration in all twenty-one specimens, cysts in eight, and fibrosis in ten. In addition, we examined 368 autopsy specimens from people who had been between twenty-five and eighty-five years old at the time of death. In many of the subjects who had died in the sixth decade of life or later, we found that the orientation of the inner fiber bundles of the anulus fibrosus was reversed, so that they bulged inward. The reversal appeared to be the result of myxomatous degeneration of the middle fibers of the anulus, atrophy of the nucleus, and narrowing of the disc space. These histological findings suggest explanations for the predominance of protrusions of the nucleus pulposus in patients who are less than sixty years old and of prolapse of the anulus fibrosus in the few patients who are more than sixty years old who have herniation of an intervertebral disc.     

Histology of intervertebral disc protrusion: an experimental study using an aged rat model

STUDY DESIGN: In vitro experimental intervertebral disc ruptures of aged rats were examined histologically. OBJECTIVES: To clarify the mechanism of intervertebral disc herniations by microscopic investigation of ruptured discs. SUMMARY OF BACKGROUND DATA: Clinically, disc herniations have been classified into two types: extrusion and protrusion. However, the pathogenesis of protrusion type herniations has not yet been demonstrated by any studies. To clarify this issue, it is essential to establish an appropriate model producing disc herniations, and to examine the sequential changes in the structure of herniated discs. METHODS: Lumbar discs of 2-year-old rats were examined histologically and compared with human lumbar discs. To examine structural changes in discs subjected to repetitive motion stress, 400 repetitions of a sequence of flexion (30 degrees ) and axial rotation (6 degrees ) were applied in vitro to the lumbar discs of the animals. RESULTS: The microstructure of normal lumbar discs in aged rats was similar in many ways to the human lumbar discs in a 20- to 40-year-old adult. Of 10 discs subjected to repetitive stress, 4 were ruptured at the junction between the posterior anulus fibrosus and the sacral cartilage endplate. One had an extruded nucleus pulposus, and three had a protruded anulus fibrosus, which displayed disorganized structure containing widened and flaccid lamellae. CONCLUSIONS: The results from this study indicate that disc protrusion can be caused by disorganization of the ruptured annular lamellae, not by focal compression of the nucleus pulposus.     

Age changes in lumbar vertebrae and intervertebral discs

Reduction of stature in old age has been attributed to loss of disc height. A measurement study of 204 cadaveric lumbar spines from subjects ranging in age from one day to 97 years confirmed data on loss of "spinal stature" but could not confirm a loss of disc height. With aging, there is a progressive increase in vertebral end-plate concavity, associated with decreased bone density. These changes are more evident and take place earlier in females than in males. In the cancellous bone of vertebral bodies, a decrease in the number of horizontal trabecular "cross braces" leads to fracture of the vertical weight-bearing "beams" supporting the vertebral endplate. The intervertebral discs expand centrally and become increasingly convex. Measurements of average disc height demonstrate that loss of disc height is unusual in a normal, aging population. Only a minority of lower lumbar discs from elderly subjects show "thinning" and degeneration (beyond Rolander's Grade 2). Thus, loss of stature in the elderly is attributable to loss in vertebral height rather than loss in disc height. Dessication and thinning of discs, or discs that "bulge like underinflated automobile tires" are not typical of elderly spines.     

Stress in lumbar intervertebral discs during distraction: a cadaveric study

Background contextThe intervertebral disc is a common source of low back pain (LBP). Prospective studies suggest that treatments that intermittently distract the disc might be beneficial for chronic LBP. Although the potential exists for distraction therapies to affect the disc biomechanically, their effect on intradiscal stress is debated.PurposeTo determine if distraction alone, distraction combined with flexion, or distraction combined with extension can reduce nucleus pulposus pressure and posterior annulus compressive stress in cadaveric lumbar discs compared with simulated standing or lying.Study designLaboratory study using single cadaveric motion segments.Outcome measuresStrain gauge measures of nucleus pulposus pressure and compressive stress in the anterior and posterior annulus fibrosus.MethodsIntradiscal stress profilometry was performed on 15 motion segments during 5 simulated conditions: standing, lying, and 3 distracted conditions. Disc degeneration was graded by inspection from 1 (normal) to 4 (severe degeneration).ResultsAll distraction conditions markedly reduced nucleus pressure compared with either simulated standing or lying. There was no difference between distraction with flexion and distraction with extension in regard to posterior annulus compressive stress. Discs with little or no degeneration appeared to distribute compressive stress differently than those with moderate or severe degeneration.ConclusionsDistraction appears to predictably reduce nucleus pulposus pressure. The effect of distraction therapy on the distribution of compressive stress may be dependent in part on the health of the disc.     

Regeneration of intervertebral discs in a rat disc degeneration model by implanted adipose-tissue-derived stromal cells

Background  

Because adipose-tissue-derived stromal cell (ADSC) is readily accessible and abundant in stem cell, ADSC may be a better candidate for cell therapy and tissue engineering. This study investigated the potential of ADSC implantation to restore disc in a rat IVD model.     

Serial changes of herniated intervertebral discs after posterior lumbar discectomy: the relation between magnetic resonance imaging of the postoperative intervertebral discs and clinical outcome

To evaluate the relation between the morphologic changes of postoperative intervertebral discs and the clinical outcome after posterior lumbar discectomy, the size of the bulging disc was analyzed prospectively on serial follow-up magnetic resonance images in 26 randomly selected patients. The bulging of postoperative intervertebral discs involved three patterns of reduction: early reduction (n = 15), gradual reduction (n = 6), and late reduction (n = 5). There was a significant difference in the serial changes of subjective symptoms and neurologic function among the three patterns. A late reduction of postoperative disc bulging could cause late recovery of subjective symptoms and neurologic disturbance.     

Histological changes in rat testicles after short-term ischaemia

A temporary pinching off of the spermatic cord was carried out in 100 male Wistar rats in order to evaluate the effect of a limited period of ischaemia on the testicular parenchyma. The animals were divided into four groups of equal number, and a certain period of ischaemia was chosen (15, 30, 45 and 60 min). The histological investigation of the testicle parenchyma was performed 2 months after the operation, the contralateral testicle serving as control. Already an ischaemia of 15 min leads to a statistically significant, irreversible damage of 53.5% of the seminiferous tubulus (p = 0.001). The degree of the damage does not increase up to an ischaemic period of 1 h. Since rat testicles have proven to be a model for the judgement of human fertility, operations on testicles which are combined with a temporary ischaemia of this organ, should be ended within a few minutes.     

Accelerated aging of intervertebral discs in a mouse model of progeria

Intervertebral disc degeneration (IDD) is a common and debilitating disorder that results in reduced flexibility of the spine, pain, and reduced mobility. Risk factors for IDD include age, genetic predisposition, injury, and other environmental factors such as smoking. Loss of proteoglycans (PGs) contributes to IDD with advancing age. Currently there is a lack of a model for rapid investigation of disc aging and evaluation of therapeutic interventions. Here we examined progression of disc aging in a murine model of a human progeroid syndrome caused by deficiency of the DNA repair endonuclease, ERCC1–XPF (Ercc1^?/Δ mice). The ERCC1‐deficient mice showed loss of disc height and degenerative structural changes in their vertebral bodies similar to those reported for old rodents. Compared to their wild‐type littermates, Ercc1^?/Δ mice also exhibit other age‐related IDD characteristics, including premature loss of disc PG, reduced matrix PG synthesis, and enhanced apoptosis and cell senescence. Finally, the onset of age‐associated disc pathologies was further accelerated in Ercc1^?/Δ mice following chronic treatment with the chemotherapeutic agent mechlorethamine. These results demonstrate that Ercc1^?/Δ mice represent an accurate and rapid model of disc aging and provide novel evidence that DNA damage negatively impacts PG synthesis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1600–1607, 2010     

Age-related changes in fibromodulin and lumican in human intervertebral discs.

STUDY DESIGN: An analysis of proteoglycans of the intervertebral disc using immunoblotting of tissue extracts. OBJECTIVES: To investigate the changes in structure and abundance of fibromodulin and lumican in human intervertebral discs during aging and degeneration. SUMMARY OF BACKGROUND DATA: Fibromodulin and lumican are keratan sulfate proteoglycan constituents of the disc's extracellular matrix, whose interaction with collagen fibrils may contribute to the mechanical properties of the tissue. Changes in their abundance and/or structure that occur with aging and degeneration therefore may have an impact on disc function. METHODS: Lumbar intervertebral discs were obtained from individuals of different ages, and extracts of anulus fibrosus and nucleus pulposus were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for fibromodulin and lumican. RESULTS: The major changes in abundance observed with age were a decrease in fibromodulin in the adult nucleus pulposus and an increase in lumican in anulus fibrosus during early juvenile development. In addition, fibromodulin in the anulus fibrosus exhibited a structural change with increasing age, characterized by a shift toward the predominance of its glycoprotein form lacking keratan sulfate. Fibromodulin was more abundant in the anulus fibrosus than in nucleus pulposus at all ages, whereas lumican was much more abundant in nucleus pulposus than in anulus fibrosus in the young juvenile; in the adult, however, lumican was present in comparable levels in both tissues. With increasing degrees of degeneration, fibromodulin exhibited an increase in abundance. CONCLUSIONS: Growth, aging, and degeneration of the intervertebral disc are associated with changes in the abundance and structure of fibromodulin and lumican, which presumably influence the functional properties of the tissue.     

大鼠退变腰椎间盘组织的超微结构观察

[目的]观察大鼠腰椎间盘退变动物模型中椎间盘组织的超微结构改变。[方法]32只SD大鼠随机分为实验组和对照组，每组各16只。实验组采用手术方法以L3为中心切除棘突、关节突、棘上、棘间韧带，切断双侧竖棘肌。对照组仅切开皮肤后即缝合。术后8周，应用电镜技术对SD大鼠椎间盘组织进行详细的超微结构观察。[结果]对照组的椎间盘组织超微结构的病理改变不显著，而实验组表现为软骨样细胞减少，出现不同程度地退变、坏死，细胞器数目减少，细胞外周致密颗粒增多；基质中胶原纤维发生不同程度的变性、融合、扭结或钙化，胶原纤维束间裂隙增大；[结论]应用电镜观察腰椎间盘退变动物模型的超微病理改变，可为研究腰椎间盘疾患提供相关的实验依据。     

Histological study of muscle in the experimental hypophosphatemic rat

Using hypophosphatemic rat maintained on a low phosphate diet as a model for human rickets and osteomalacia, the skeletal muscle was histochemically and electron microscopically examined, in comparison with specimens obtained from normal control animals. In muscles obtained from hypophosphatemic animals, the type 2 muscle fibers were increased in number, and the type 1 fibers were atrophic. Electron microscopic studies revealed some atypical structures of mitochondria with fusion of cristae in the muscle spindles of intrafusal muscle fibers. The presynapse, the secondary cleft, and the endplate plasm were significantly atrophic in the extrafusal neuromuscular junction. Mitochondrial vacuolization was observed in the presynapse. These results indicated the presence of immature muscle fibers, dysfunction of the energy metabolism of the mitochondria and neurogenic disorders in hypophosphatemic muscles.     

Relationship between neovascularization and degenerative changes in herniated lumbar intervertebral discs

Purpose

Lumbar disc degeneration may be associated with intensity of neovascularization in disc herniations. Our study was designed to evaluate how much the severity of histodegeneration is related to the development of neovascularization and to the level of pleiotrophin in the herniated lumbar discs.

Methods

Surgically excised lumbar disc specimens were obtained from 29 patients with noncontained (i.e., extruding through the posterior longitudinal ligament) and 21 patients with contained disc herniations. The histodegeneration scores and levels of neovascularization were estimated according to semiquantitative analysis in lumbar disc and endplate samples. Immunohistochemical staining were performed to identify the newly formed blood vessels and to detect the presence of pleiotrophin in the specimens.

Results

Higher levels of disc and endplate neovascularity were registered in noncontained herniations. The level of neovascularization was significantly related to the score of histodegeneration in the herniated disc tissues but not in the endplate specimens. Both contained and noncontained herniations had the highest values of histodegeneration in conjunction with the highest level of neovascularization but the relations between neovascularity and degenerative changes remained to be significant only in the group of noncontained herniations. Registration or frequency of pleiotrophin positive cells did not correlate significantly with histodegeneration or level of neovascularization in the disc samples.

Conclusion

Severe histodegeneration of the lumbar disc herniations is associated with enhanced neovascularization and potentially also spontaneous regression of the herniated tissue.     

Height change caused by creep in intervertebral discs: a sagittal plane model.

Changes in spinal column height have been observed in response to different stress environments including vibration, gravity inversion, space flight, traction, and increased loading. Alterations in spinal height are dependent on body forces, externally applied forces, and properties of the discs and are considered relevant to understanding the normal and pathologic behavior of the spine. This study presents a sagittal plane, viscoelastic model of the spine that quantified the height change behavior of the human spine subjected to axial compressive forces similar to those experienced during quiet standing. The two-dimensional spine model was idealized as a collection of 23 rigid vertebral bodies and 23 deformable intervertebral discs. Time-dependent height losses were modeled using axial compressive creep material properties based on in vitro measurements obtained from the literature. The model demonstrated an instantaneous loss in height of 11.7 mm (0.67% of body height) and a height loss of 19.6 mm (1.1% of body height) at the end of 8 h. Changes in sagittal profile were estimated to contribute to 12% of the overall height loss after 8 hours. Discs in the lumbar region lost the most height, but the contribution of the lumbar region to the total height loss was 32%. The height loss contribution of the thoracic region was higher (57%), presumably because of the increased number of discs contributing to the total height loss in this region. For degenerated discs, the model predicted a similar instantaneous height loss but a 28% greater height loss after 8 h. These results suggest that the majority of spinal height loss is a direct result of intervertebral disc deformation and about two thirds of the total height loss occurs immediately on axial loading of the spine. Based on these findings, diurnal height changes in the spine are predicted to be much greater than previously believed.